For the past several years, we have been investigating the role of latently infected, resting CD4+ T cells and persistent low level viral replication in the pathogenesis of HIV infection, and the impact of viral reservoirs in HIV-infected individuals receiving antiretroviral therapy (ART). We previously demonstrated that the latent viral reservoir in the resting CD4+ T cell compartment persists in virtually all HIV-infected individuals receiving effective ART. Consequently, this viral reservoir is a major impediment to the eradication of HIV in vivo. In addition, we demonstrated that HIV continually replicates at low levels in chronically infected individuals who are consistently aviremic during prolonged periods of receiving ART. During the past year, we have focused our research on 1) delineating the mechanisms by which HIV persists in infected individuals receiving ART for extended periods of time, and 2) investigating the dynamics of decay of viral reservoirs in HIV-infected individuals who initiate ART at different stages of disease. First, we examined the HIV burden in a subset of resting CD4+ T cells expressing Programmed Death (PD)-1 in infected individuals receiving ART. It has been proposed that the HIV present in infected resting CD4+ T cells is immunologically and virologically quiescent. We demonstrated that resting CD4+ T cells that express PD-1 carried substantially higher levels of HIV proviral DNA compared with PD-1-negative resting CD4+ T cells in the blood of HIV-infected individuals receiving effective ART (viral load below detectable level, i.e. <50 copies HIV RNA/mL). The majority of PD-1+ resting CD4+ T cells expressed CXCR3, a tissue-homing receptor, suggesting that these cells may have recently migrated out of various tissue sites into the peripheral blood. PD-1+ resting CD4+ T cells from the majority of patients receiving clinically successful ART spontaneously released HIV in the absence of any activating stimuli. The pattern of mRNA expression involving cellular activation and cytokine and chemokine genes in PD-1+ resting CD4+ T cells also was distinct from that of PD-1-negative resting and activated CD4+ T cells in the study participants. Taken together, our data suggest that PD-1+ resting CD4+ T cells represent a unique population of infected cells that persist during effective ART in HIV-infected individuals. Second, we investigated the dynamics of decay of viral reservoirs in HIV-infected individuals who initiated ART at different stages of disease. In a cross-sectional study, we demonstrated that the median copy number of HIV proviral DNA in patients who had initiated ART within 6 months of infection was significantly lower compared to patients who had initiated ART during the chronic phase of infection (p=0.003). To examine the frequency of CD4+ T cells carrying infectious virus, a High-Input Co-Culture assay, which allows examination of large numbers of cells, was conducted using highly enriched CD4+ T cells from the eight HIV-infected individuals in whom no measurable HIV proviral DNA had been detected. The frequency of cells carrying infectious virus in HIV-infected individuals who initiated therapy within 6 months of infection was significantly lower than that of HIV-infected individuals who initiated therapy during the chronic phase of infection (p=0.03). Two HIV-infected individuals, one in whom ART was initiated during chronic infection and one in whom ART was initiated during the early phase of infection underwent colonoscopies to measure the levels of HIV in gut-associated lymphoid tissue (GALT). Real-time PCR was performed on CD8-depleted cells isolated from multiple sigmoid colon biopsies from each patient. Although HIV proviral DNA was undetectable in peripheral blood CD4+ T cells in the patient in whom therapy was initiated during chronic HIV infection, infectious virus was recovered and PCR revealed readily detectable HIV DNA in GALT. The HIV-infected individual who initiated ART during the early phase of infection similarly had undetectable HIV proviral DNA, but the level of infectious virus was extraordinarily low (one infected cell per 1.7x10e9 CD4+ peripheral blood T cells), and HIV DNA was undetectable in CD8-depleted cells isolated from GALT. These results suggest that a profound reduction in the size of viral reservoir occurred in the individual receiving ART during the early stages of HIV infection. To investigate whether temporarily ceasing therapy in this individual with an extraordinarily low HIV reservoir would result in a rebound of plasma viremia, we received the patients consent to safely discontinue ART and carefully monitor changes in plasma viremia. Plasma viremia was not detected for the first 50 days after therapy interruption. Subsequently, plasma viremia rebounded to 1,593 copies of HIV RNA per milliliter (ml), followed by spontaneous suppression to an undetectable level. Plasma viremia rebounded again to 8,684 copies of HIV RNA per ml on day 143, ART was resumed at this time. Our data clearly suggest that, at least in a subset of HIV-infected individuals, the profound suppression of viral replication by long-term, effective ART initiated early during the course of infection may lead to a substantially greater reduction of residual HIV compared with that observed in individuals who initiated ART during the chronic phase of infection. Unfortunately, our study also demonstrated that the combination of early initiation of ART, an extended duration of therapy, and a profoundly low HIV burden in CD4+ T cells did not eradicate HIV, nor did it indefinitely suppress the re-emergence of plasma viremia. However, contrary to previous reports, this combination of early initiation and long duration of therapy did lead to a longer period (50 days) of aviremia after the interruption of antiretroviral drugs (average 9 days). To achieve a condition under which HIV does not rebound for extended periods of time in the absence of ART, novel therapeutic strategies specifically targeting these extremely rare infected cells may be necessary, with or without the use of therapeutic vaccination to boost immune control of viral rebound.